A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis.

Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

Development of an Effective Nontoxigenic Clostridioides difficile-Based Oral Vaccine against C. difficile Infection.

The symptoms of Clostridioides difficile infection (CDI) are largely attributed to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. Recently, we generated a novel chimeric protein (designated Tcd169), comprised of the glucosyltransferase domain (GT), the cysteine protease domain (CPD), and the receptor binding domain (RBD) of TcdB, and the RBD of TcdA. Parenteral immunizations with Tcd169 provide mice effective protection against infection with a ribotype (RT) 027 C. difficile strain. In this study, we expressed Tcd169 in a nontoxigenic C. difficile CCUG37785 strain (designated NTCD), resulting in strain NTCD_Tcd169 to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors. Oral immunizations with NTCD_Tcd169 spores induced systematic and mucosal antibody responses against, not only both toxins, but also C. difficile flagellins (FliC/FliD). Intriguingly yet importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2). Oral immunizations with NTCD_Tcd169 spores provided mice effective protection against infection with a hypervirulent RT027 C. difficile strain R20291and significantly reduced R20291spore numbers in feces compared with NTCD or PBS immunized mice. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing Tcd169 may represent a novel mucosal vaccine candidate against CDI. IMPORTANCE Clostridioides difficile is an enteric pathogen, and symptoms of C. difficile infection (CDI) are mainly by two exotoxins TcdA and TcdB. Active vaccination is cost-effective approach to prevent CDI and high rates of recurrence. Ideally, vaccines should target both C. difficile toxins and cell/spore colonization. In this study, we expressed immunodominant fragments of TcdA and TcdB (i.e., Tcd169) in a nontoxigenic C. difficile CCUG37785 strain, generating a promising oral/mucosal vaccine candidate against CDI, by targeting both toxins and colonization of pathogenic C. difficile strains. Importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2), and all of which are involved in C. difficile adhesion/colonization in vitro and in vivo.

FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291.

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism "FliW-CsrA-Hag." In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of "FliW-CsrA-Hag" switch coregulation mechanism reported in B. subtilis, our data also suggest that "FliW-CsrA-fliC/FliC" can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile.

Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

Effective adsorption of nisin on the surface of polystyrene using hydrophobin HGFI.

Herein, a new method was demonstrated for effective immobilization of the antibacterial peptide nisin on Grifola frondosa hydrophobin (HGFI), without the need of any additional complex reaction. Hydrophobin can self-assemble as a monolayer to form continuous negative-charged surfaces with enhanced wettability and biocompatibility. Adding nisin solution to such hydrophobin surface created antibacterial surfaces. The quantification analysis revealed that more nisin could be adsorbed on the HGFI-coated than to control polystyrene surfaces at different pH values. This suggested that electronic attraction and wettability may play important roles in this process. The transmission electron microscopy, atomic force microscopy and fourier transform infrared (FTIR) analysis indicated the adsorption mode of nisin on the HGFI film, i.e., hydrophobins served as an adhesive layer for binding charged peptides to interfaces. The antibacterial activity of the treated surface was investigated via counting, a nucleic acid release test, scanning electron microscopy, and biofilm detection. These results indicated the excellent antibacterial activity of nisin adsorbed on the HGFI-coated surfaces. The activity retention of adsorbed nisin was demonstrated by immersing the modified substrates in a flowed liquid condition.

Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system.

BACKGROUND: After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. RESULTS: Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. CONCLUSIONS: Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.

Characterization of the virulence of a non-RT027, non-RT078 and binary toxin-positive Clostridium difficile strain associated with severe diarrhea.

The expression of the Clostridium difficile binary toxin CDT is generally observed in the RT027 (ST1) and RT078 (ST11) C. difficile isolates, which are associated with severe C. difficile infection (CDI). However, we recently reported that the non-RT027 and non-RT078 C. difficile strain LC693 (TcdA+TcdB+ CDT+, ST201) caused severe diarrhea in a patient in Xiangya Hospital in China. C.difficile LC693 is a member of Clade 3, and in this study, we identified LC693 as RT871 and compared its virulence and pathogenicity to those of C.difficile R20291 (TcdA+TcdB+CDT+, ST1/RT027), UK6 (TcdA+TcdB+CDT+, ST35/RT027), CD630 (TcdA+TcdB+CDT-, ST54, RT012), and 1379 (TcdA+TcdB+CDT-, ST54/RT012), with strain 1379 being an epidemic C.difficile isolate from the same hospital. LC693 displayed a higher sporulation rate than R20291, CD630 or strain 1379. LC693 was comparable to R20291 with respect to spore germination, motility, and biofilm formation, but showed a faster germination rate, higher motility and a higher biofilm formation capability compared to CD630 and strain 1379. The adherence of spores to human gut epithelial cells was similar for all strains.The total toxin release of LC693 was lower than that of R20291, but higher than that of CD630 and strain 1379. Finally, in a mouse model of CDI, LC693 was capable of causing moderate to severe disease. Our findings demonstrate the pathogenicity of non-RT027 and non-RT078 binary toxin-positive C. difficile strains. Furthermore, our data indicate that LC693 may be more virulent than strain 1379, an epidemic strain from the same hospital, and provide the first phenotypic characterization of a non-RT027 and non-RT078 binary toxin-positive ST201 isolate.

Bioprospecting Deep-Sea Actinobacteria for Novel Anti-infective Natural Products.

The global prevalence of drug resistance has created an urgent need for the discovery of novel anti-infective drugs. The major source of antibiotics in current clinical practice is terrestrial actinobacteria; the less-exploited deep-sea actinobacteria may serve as an unprecedented source of novel natural products. In this study, we evaluated 50 actinobacteria strains derived from diverse deep water sponges and environmental niches for their anti-microbial activities against a panel of pathogens including Candida albicans, Clostridium difficile, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa. More than half of the tested strains (27) were identified as active in at least one assay. The rare earth salt lanthanum chloride (LaCl3) was shown to be as an effective elicitor. Among the 27 strains, the anti-microbial activity of 15 were induced or enhanced by the addition of LaCl3. This part of study focused on one strain R818, in which potent antifungal activity was induced by the addition of LaCl3. We found that the LaCl3-activated metabolites in R818 are likely antimycin-type compounds. One of them, compound 1, has been purified. Spectroscopic analyses including HR-MS and 1D NMR indicated that this compound is urauchimycin D. The antifungal activity of compound 1 was confirmed with a minimal inhibitory concentration (MIC) of 25 µg/mL; the purified compound also showed a moderate activity against C. difficile. Additional notable strains are: strain N217 which showed both antifungal and antibacterial (including P. aeruginosa) activities and strain M864 which showed potent activity against C. difficile with an MIC value (0.125 µg/mL) lower than those of vancomycin and metronidazole. Our preliminary studies show that deep-sea actinobacteria is a promising source of anti-infective natural products.

Co-expression of Nisin Z and Leucocin C as a Basis for Effective Protection Against Listeria monocytogenes in Pasteurized Milk.

Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin-leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.

Clostridioides difficile Biology: Sporulation, Germination, and Corresponding Therapies for C. difficile Infection.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe, and an important nosocomial pathogen. Due to the strictly anaerobic nature of the vegetative form, spores are the main morphotype of infection and transmission of the disease. Spore formation and their subsequent germination play critical roles in C. difficile infection (CDI) progress. Under suitable conditions, C. difficile spores will germinate and outgrow to produce the pathogenic vegetative form. During CDI, C. difficile produces toxins (TcdA and TcdB) that are required to initiate the disease. Meanwhile, it also produces spores that are responsible for the persistence and recurrence of C. difficile in patients. Recent studies have shed light on the regulatory mechanisms of C. difficile sporulation and germination. This review is to summarize recent advances on the regulation of sporulation/germination in C. difficile and the corresponding therapeutic strategies that are aimed at these important processes.

A Novel Bacteriophage Lysin-Human Defensin Fusion Protein Is Effective in Treatment of Clostridioides difficile Infection in Mice.

Clostridioides difficile is the leading cause of worldwide antibiotics-associated diarrhea. In this study, we report the construction and evaluation of a novel bacteriophage lysin-human defensin fusion protein targeting C. difficile. The fusion protein, designated LHD, is composed of two parts connected by a 3-repeating unit linker "(GGGGS)3": the catalytic domain of a lysin protein from a C. difficile bacteriophage phiC2 (LCD), and the functional domain of a human defensin protein HD5. Lytic assays showed that LHD protein had a potent lytic activity against different types of clinical C. difficile strains, including the epidemic 027, 078, 012, and 087 strains. The minimum inhibitory concentration (MIC) of LHD was 0.78 µg/ml, which was lower than the MIC of the protein LCD (1.56 µg/ml), and the MICs of metronidazole (4 µg/ml) and vancomycin (4 µg/ml). In addition, the LHD protein could lyse C. different strains in different pHs (6.0, 7.0, and 8.0). Evaluation of LHD potency in vivo using mouse model of C. difficile infection (CDI) showed that administration of the LHD protein (twice daily for 7 days) was effective in mitigating the symptoms and reducing the death from CDI. Treatment with LHD also significantly decreased the number of C. difficile spores and the toxin level in feces from the infected mice. Our data suggest that this novel lysin-human defensin fusion protein has a potential on CDI control.

Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.

BACKGROUND: The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation. RESULTS: Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly. CONCLUSIONS: The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.

[Effect of 6-phosphofructokinase gene-pfk overexpression on nisin production in Lactococcus lactis N8].

OBJECTIVE: To accelerate the formation of nisin through overexpressing 6-phosphofructokinase gene pfk in nisin-producer Lactococcus lactis N8. METHODS: The genes of pfk and pkaC encoding the catalytic subunit of cAMP-dependent protein kinase were cloned into the vector pMG36e andtransformed into L. lactis N8, resulting in the recombinant strain L. lactis N8-pMG36e-pfk-pkaC. Several biochemical and physological factors, including growth profiles, activity of 6-phosphofructokinase, expression of nisA , antibacterial activity of supernatants and nisin titer, were monitored to investigate the differences between the recombinant strain and the parental strain. RESULTS: No significant difference was observed with respect to the growth patterns of the recombinant strain and the wild type. As expected, the biological activity of PFK in recombinant strain was increased for all examined samples. Correspondingly, the yield of nisin was increased by 20% in the recombinant strain after fermentation for 10 hours, which could be attributed to the accelerated biosynthesis of nisin. As a result, the fermentation cycle was reduced about 2 hours. Meanwhile, different concentration of glucose did not affect the formation of nisin. CONCLUSION: The overexpression of pfk and pkaC genes in the nisin-producer strain can effectively accelerate nisin biosynthesis.

Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter is commonly used for induced expression of proteins in L. lactis, whereas few constitutive promoters (P45 and the weaker P32) have been used for protein expression studies. In this study, eight different putative strong constitutive promoters were identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3 and P2) were identified as having a transcriptional activity that was higher than that of P45 through RT-qPCR and agar-diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene (ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. The transcriptional efficiencies of promoters P8, P5, P2 and P3 were all higher than promoter P45 based on the obtained MIC50 values and they all showed different activity levels. In conclusion, four strong constitutive promoters with a wide range of promoter activities were identified and are suitable for protein production in L. lactis.